Catalytic reaction profile for alcohol oxidation by galactose oxidase

Galactose oxidase is a remarkable enzyme containing a metalloradical redox cofactor capable of oxidizing a variety of primary alcohols during enzyme t urnover. Recent studies using 1-O-methyl alpha -D-galactopyranoside have re vealed an unusually large kinetic isotope effect (KIE) for oxidation of the alpha -deuterated alcohol (k(H)/k(D) = 22), demonstrating that cleavage of the 6,6'-di[H-2]hydroxymethylene C-H bond is fully rate-limiting for oxida tion of the canonical substrate. This step is believed to involve hydrogen atom transfer to the tyrosyl phenoxyl in a radical redox mechanism for cata lysis [Whittaker, M. M., Ballou, D. P., and Whittaker, J. W. (1998) Biochem istry 37, 8426-8436]. In the work presented here, the enzyme's unusually br oad substrate specificity has allowed us to extend these investigations to a homologous series of benzyl alcohol derivatives, in which remote (meta or para) substituents are used to systematically perturb the properties of th e hydroxyl group undergoing oxidation. Quantitative structure-activity rela tionship (QSAR) correlations over the steady state rate data reveal a shift in the character of the transition state for substrate oxidation over this series, reflected in a change in the magnitude of the observed KIE for the se reactions. The observed KIE values have been shown to obey a log-linear correlation over the substituent parameter, Hammett sigma. For the relative ly difficult to oxidize nitro derivative, the KIE is large (k(H)/k(D) = 12. 3), implying rate-limiting C-H bond cleavage for the oxidation reaction. Th is contribution becomes less important for more easily oxidized substrates (e.g., methoxy derivatives) where a much smaller KIE is observed (k(H)/k(D) = 3.6). Conversely, the solvent deuterium KIE is vanishingly small for 4-n itrobenzyl alcohol, but becomes significant for the 4-methoxy derivative (k (H2O)/k(D2O) = 1.2) These experiments have allowed us to develop a reaction profile for substrate oxidation by galactose oxidase, consisting of three components (hydroxylic proton transfer, electron transfer, and hydrogen ato m transfer) comprising a single-step proton-coupled electron transfer mecha nism. Each component exhibits a distinct substituent and isotope sensitivit y, allowing them to be identified kinetically. The proton transfer componen t is unique in being sensitive to the isotopic character of the solvent (H2 O or D2O), while hydrogen atom transfer (C-H bond cleavage) is independent of solvent composition but is sensitive to substrate labeling. In contrast, electron transfer processes will in general be less sensitive to isotopic substitution. Our results support a mechanism in which initial proton abstr action from a coordinated substrate activates the alcohol toward inner sphe re electron transfer to the Cu(II) metal center in an unfavorable redox equ ilibrium, forming an alkoxy radical which undergoes hydrogen atom abstracti on by the tyrosine-cysteine phenoxyl free radical ligand to form the produc t aldehyde.